3.1 Characterization of CDH and CHMO
CDH and CHMO were cloned separately into the pSEVA244_T vector[18] to characterize their in vivoactivity. As CDH catalyzes an equilibrium reaction, the kinetic
parameters were assayed for both reaction directions (Table 1). For the
reverse reaction with cyclohexanone as substrate, CDH showed a 10 times
lower Vmax compared to the forward reaction. On the
other hand, the KS values differed by a factor of almost
100 in favor of the reverse reaction (0.05 and 3.57 mM for cyclohexanol
and cyclohexanone, respectively). Furthermore, we theoretically and
experimentally assessed the cyclohexanol/cyclohexanone concentration
ratio at equilibrium. Utilizing the group contribution method[24] assuming a physiological intracellular NADH
to NAD concentration ratio of 10.6 under aerobic conditions[25], this ratio was determined to be 1.9
(Supplementary Information, Section 4). It was confirmed experimentally
by applying different initial alcohol and ketone concentrations giving a
cyclohexanol/cyclohexanone concentration ratio of 1.95 ± 0.29 after 16 h
(Figure S2). This thermodynamic preference of the backward reaction,
together with the low KS value for cyclohexanone
emphasizes the necessity of an efficient cyclohexanone withdrawal by the
successive enzyme in the cascade, i.e., CHMO.
Substantial research has been conducted with a cyclohexanone
monooxygenase originating from Acinetobacter sp.[26]. Generally, the substrate as well as product
toxicity, are features of Baeyer-Villiger monooxygenase-catalyzed
reactions [22, 23]. Substrate toxicity was
generally observed at aqueous concentrations in the mM-range, which
should thus be avoided during the cascade reaction. Furthermore, CHMO
may be inhibited by the cascade intermediate cyclohexanol and its
product ε-CL. Acidovorax CHMO indeed was found to be highly prone
to inhibition by cyclohexanol (Figure 2A). At a cyclohexanol
concentration as low as 0.4 mM, the high initial CHMO activity of
160.3 ± 0.1 U gCDW-1 was found to be
reduced to half this rate. Cyclohexanol concentrations ≥ 1.7 mM
completely abolished CHMO activity. However, up to an ε-CL concentration
of 17 mM, no product inhibition was found for CHMO (Figure 2B).
Summing up, these results emphasize that the produced cyclohexanol needs
to be directly converted by CDH to avoid CHMO inhibition. High
intracellular CDH and CHMO activities are important to avoid any
accumulation of alcohol and ketone intermediates, as already low alcohol
amounts can be expected to inherently reinforce such accumulation.